1. Field of the Invention.
The present invention relates to an apparatus and method for qualitative and/or quantitative analysis of a specific binding complex and to a system for interpreting the shadows created on a detector by light passing through the specific binding complex.
2. Description of the Prior Art.
biological systems involve the specific binding of two independent reactants to form a specific binding complex (SBC). These reactants have been the basis of assay systems which attempt to determine the presence of the unknown reactant by using a known amount of a specific receptor chemical (SRC) in a variety of configurations. Most of these assays are based on labeling the SRC with detectable substances such as radioisotopes, enzymes, fluorochromes and the like. The labeled SRC is detected, for example, by use of a scintillation counter after the radioactively labeled SRC and the unknown reactant have been allowed to form the SBC.
Typical of the described assays are immunoassays, which employ a labeled specific antibody to detect the presence of its antigen and, in some cases, quantify the amount of antigen present in the test sample. Examples of such assays are found in Zuk and Ullman U.S. Pat. No. 4,256,834 and Hornby et.al. U.S. Pat. No. 4,238,565 as well as in Weir, Handbook of Experimental Immunology, 3rd ed., Blackwell Scientific Publications, Philadelphia, PA.
In these prior art systems, the formation of the SBC and its detection are accomplished on a glass or plastic surface, or in a container. After the SBC is formed, the net reaction in the system is measured. The problem encountered in such prior art systems is that the reactants need to come to equilibrium (which usually takes 30 to 120 minutes) to form a sufficient number of SBCs for detection by the system. After equilibration, the level of the SBCs formed during the reaction is estimated by measuring the quantity of labeled SRC bound to the unknown reactant on the solid phase or in the container. The value obtained is then compared to a standard or independent control test to determine if a particular reactant is present and, in some cases, to provide an estimate of the unknown reactant's concentration.
Biological reagents are inherently variable. Therefore, values obtained from experimental controls will vary from reagent lot to reagent lot, and from test to test with the same lot of reagents. This variation can lead to large differences in result interpretation with respect to the amount of unknown reactant in any one test. Also, the prior art systems require the independent assay of standard(s) or control samples since they cannot be incorporated with the method or apparatus. Use of standardized reagents reduces the variation between the unknown reactant assay and the independently run control assay. However, the reduction is not sufficient to provide the type of accurate assay results that are presently needed.
Some prior art systems employ lasers in the detection of products of a specific binding reaction by concentrating on the detection of the total label bound at equilibrium, or a change in some related factor after the reaction has come to equilibrium. For example, Uzgiris U.S. Pat. No. 4,171,956 discloses a method wherein a sample is vaporized, a laser exciting a fluorescent molecule bound to one of the specific reactants in the vaporized test sample. Light is emitted by the fluorescent molecule. Another example is that described in Noeller U.S. Pat. No. 4,419,483 wherein polarized laser light stimulates the fluorescent label attached to one of the specific reactants in the reaction mixture. The difference between the level of polarized fluorescent light emitted by the fluorescent label bound in the reaction and the free fluorescent label in solution is then measured.
Hirschfeld U.S. Pat. No. 4,447,546 describes a fluorescent immunoassay apparatus and method for detection of an antigen-antibody complex. Hirschfeld employs a capillary tube with one of the reactants bound to its surface. A fluorescent label on another of the specific reactants is excited by radiation from an axially disposed optical fiber within a capillary tube only if it has bound to the reactant on the surface of the capillary tube. Other similar methods are described in Carter et.al. U.S. Pat. No. 4,508,532 and Lundstrom et.al. U.S. Pat. No. 4,521,522 which show methods for measuring the effects of specific binding reactants on elliptical polarization or refraction of a source light.
In Takekawa Pat. No. 4,452,759, a light blocking system is used to measure an agglutination pattern of blood corpuscles. The light blocking system measures the presence, absence or degree of agglutination by a measurement of the total amount of light blocked. The system of the Takekawa Patent, however, would only be suitable for assay systems where agglutination or precipitation reactions occur, and would not be suitable for accurate quantitative measurements of individual specific binding complexes.
Morris et.al. U.S. Pat. No. 3,905,767 includes a process for qualitative or quantitative analysis of antigens or antibodies by using a light source, a reflective surface and a light sensor for sensing scattered light. The antigen and antibody molecules react in a gel on the reflective surface to form a precipitate. The extent to which the light beam is scattered by the precipitate is measured. Because of the requirement for the formation of a precipitate, the Morris et.al. process produces only an estimate of the total SBCs in a sample, and has limited application.